Wound healing/plastic surgery
Homeobox Genes Hoxd3 and Hoxd8 Are Differentially Expressed in Fetal Mouse Excisional Wounds

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Background

Cell signaling pathways underlying wound repair are under extensive investigation; however, there is still a poor understanding of the mechanisms orchestrating these processes. Hox genes, which are a subgroup of homeobox genes, encode for a family of transcription factors that play a critical role in tissue migration and cell differentiation during embryogenesis and may also serve as master regulatory genes of postnatal wound repair. We have developed a fetal excisional wound healing model whereby mid-gestational wounds heal in a regenerative manner while late-gestational wounds display scar formation. We theorize that Hoxd3 and Hoxd8 will be differentially expressed in mid- and late-gestational wounds compared with normal skin.

Materials and methods

Pregnant FVB mice underwent hysterotomy at mid (E15)- or late (E18)-gestational time points, and 3-mm excisional wounds were made on the dorsum of each fetus. Wound samples (w) were collected at the site of injury as well as near wound normal skin (nwc) on the same fetus. Control (c) skin samples were also obtained from unwounded adjacent fetuses. Samples were harvested at 3 and 6 h and real-time polymerase chain reaction was performed for Hoxd3 and Hoxd8 and normalized to glyceraldehyde-3-phosphate dehydrogenase. Data were analyzed by analysis of variance with statistical significance of P < 0.05.

Results

Hoxd3 levels were increased in all of the mid-gestational groups, with a significant increase at 3 h compared with late-gestational control groups. In the 3-h time group, Hoxd8 is increased in mid-gestational wounds compared with late-gestational control skin. This is repeated in the 6-h time group, where Hoxd8 is increased in mid-gestational wounds compared with all groups. Also, Hoxd8 in the mid-gestational near wound controls is significantly greater than that in the late-gestational near wound control and control groups.

Conclusions

These data suggest that Hoxd3 is constitutively expressed in the skin of mid-gestational mice. However, Hoxd8 expression is increased in the mid-gestational wounds compared with normal control groups and late gestational wounds, suggesting that it may play a role in scarless wound repair.

Introduction

Wounds that heal by excessive or sparse healing present a significant dilemma in health care and patient well being. Various disease processes are affected by flawed wound repair, such as pulmonary and liver fibrosis, contracture scars from burns, keloid formation and pressure, and diabetic ulcers. Wound healing has been researched quite steadily for decades; however, the secondary cell signaling mechanisms underlying wound repair remain unclear. There is even less known regarding the genes that control these processes and the downstream regulation that occurs.

Neonatal and adult wound healing are characterized by inflammation, fibrosis, and scarring, whereas these features are absent in fetal wound healing [1]. In fact, the fetal neodermis after wounding is practically indistinguishable from the surrounding non-injured skin, even on a microscopic level [2, 3, 4]. The differences between adult and fetal wound repair are examined in animal models using mid- and late-gestational fetuses. The mid-gestational animals represent regenerative fetal wound healing that lacks scar formation. Late-gestational animals, on the other hand, correspond to adult wound healing with scar formation [5]. Once we are able to understand the genetic and molecular mechanisms underlying the ability of the fetal wound to regenerate in this capacity, we may apply this knowledge to the vast number of diseases affected by imperfect wound healing.

Homeobox genes encode for a large family of developmental transcription factors that have a conserved 180-base pair sequence that corresponds to a 60-amino-acid region called the homeodomain [6]. This domain is over 109 years old and is responsible for the sequence-specific DNA binding of homeobox proteins [7]. These proteins are critical in morphogenesis, tissue migration, and cell differentiation in the embryo by regulating transcription or repression of target genes [6]. These processes are similar to those important in wound healing and secondary signaling for site-directed tissue repair. Homeobox genes have also been found to play a role in wound repair and regenerative wound healing. For example, the Hoxd-11 gene was found to be up-regulated in the blastema formation of regenerating Axolotl newt forelimbs following amputation, and non-regenerating forelimbs are characterized by an up-regulation of Hoxd-8 and Hoxd-10 [8]. Hox D3 has been found to bind directly to the promoters of the integrin α5 and β3 subunits, inducing subunit expression [9, 10]. Also, Hox D3-deficient wounds of diabetic animals display a reduction in expression and deposition of Type 1 collagen [11]. Hoxd8 has been previously researched in rabbits in our laboratory and was found to be differentially expressed in fetal excisional wounds induced to contract with transforming growth factor-β3 compared with control wounds [12]. Therefore, our findings as well as those discussed in the literature suggest that homeobox genes may serve as the master genes that control downstream genes and factors responsible for orchestrating secondary signaling and wound repair. We theorize that fetal scarless and scar-forming wound repair will be associated with a differential pattern of homeobox gene expression.

Section snippets

Materials and Methods

Using a previously described fetal excisional wound healing model in accordance with Institutional Animal Care and Untilization Committee approval (#0409-3354) time-dated, pregnant FVB mice (Charles River, Wilmington, ME) underwent hysterotomy at mid (E15)- or late (E18)-gestational time points, and 3-mm excisional cutaneous wounds were made on the dorsum of each fetus [5]. Wound samples (w) were collected at the site of injury as well as near wound normal skin (nwc) on the same fetus. Control

Results

Hoxd3 levels were increased in all of the mid-gestational groups, with a significant increase at 3 h compared with late-gestational control groups. However, there did not appear to be any significant differences within the mid-gestational groups. Hoxd8, on the other hand, did show several significant differences. In the 3-h time group, Hoxd8 is increased in mid-gestational wounds compared with late-gestational control skin. This is repeated in the 6-h time group where Hoxd8 is increased in

Discussion

Fetal wound healing differs from that seen in the adult in a number of aspects. There is a sparse inflammatory response during fetal healing [13]. Collagen is deposited in a more organized and rapid fashion than the adult and has an increased type 3:1 ratio than the adult [14, 15]. It also is deposited at the fetal wound site in a reticular pattern that is indistinguishable from the surrounding tissue and has greater tensile strength than adult wounds [2, 3, 4]. In our fetal mouse excisional

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